Enlarge image Rarefaction curves showing the number of observed microbial species when the data were rarefied as a function of the number of sequence reads from the individual macroaggregates, and in the inset the data were rarefied as a function of the number of sequence reads from the whole soil. Analyses were performed on a random subsample of 1043 sequences from each sample. The upward slope of the curve in the inset indicates that additional sequencing (extending the x-axis beyond the 1043 sequences) would likely reveal the presence of far more additional species in the whole soil sample. In contrast, the relatively flat slopes of the curves for the individual aggregates indicate that we can be reasonably confident of observing only ~100-200 different species in any individual aggregate, even with additional sequencing.

Results: In the first efforts to directly link 1) soil
physical structure with microbial community composition in single aggregates,
and then 2) composition directly with bioactivity, scientists at Pacific Northwest National Laboratory and Argonne
National Laboratory studied soil macroaggregates, or clumps <1 mm in diameter.
At a
scale nearly that of a defined microbial habitat, they measured physical and bacterial community structure and microbial-derived
enzyme function to determine if the physical structure of soil aggregates
constrained the composition and function of the microbial community in a
natural soil.

Specifically, they found that while bacterial
communities in single aggregates are significantly less diverse than in whole
soil, no apparent link yet exists between the community structure and soil
physical structure. Their results appeared in Soil Biology &
Biochemistry and The ISME Journal.

Soil hosts a vast abundance and diversity of microorganisms,
making it difficult to study and understand their activities at a molecular
level. Dr. Lee Ann McCue, a PNNL senior research scientist and co-author of the
two papers, said, "Carbon is stored and stabilized in soil, but the complexity
and heterogeneity of the system, and the data we generate from it, make it
tough to attribute microbial activity to observed function."

Microbial decomposition of soil organic carbon is a critical
process where carbon is lost from soil. This level of knowledge is needed to
scale processes controlled by microbes up to the regional models used to
understand global systems. Such understanding informs land management decisions
related to nutrient/carbon cycling activity and soil health.

Why It
Matters: These studies demonstrate that analytic measurements at the
sub-millimeter soil aggregate scale now are feasible and that microbial
community composition can be examined jointly with aggregate physical structure
or enzymatic activity.

"We want to see the world a microbe sees at the scale at
which it operates," said PNNL senior research scientist Dr. Vanessa Bailey, the
research team lead, "because analysis at the right scale enables us to discover
fundamental realities of how soil works in nature."

Using size as a scaling mechanism to better link the microbial
structure and functions of individual microbes and communities thereof helps researchers understand how microbes work
within the constraints of their ecosystems.

"It may not be the presence or absence of organisms that
drive activity in soil ecosystems, but their access to, and availability of, patchily
distributed resources in the microbial environment," Bailey explained. The <1-mm
aggregate scale soil "census" information derived from these studies enables
better understanding of the processes and potentials of soil.

"The heterogeneity of soils makes it really challenging to
study," said McCue. "That's why it's so vital to gather these diverse types of
data and why it's fun to analyze and integrate."

Methods:
In
the first study, the PNNL and ANL team characterized structures of 14
individual aggregates using synchrotron radiation-based transmission X-ray microtomography
and characterized microbial community composition using high-throughput
sequence data of the 16S rRNA gene.

In the second study, PNNL scientists measured bacterial
function using scaled-down assays for adenosine triphosphate and carbon-degrading
enzymes followed by 16S gene sequencing to assess the bacterial community
structure.

The figure
shown here depicts rarefaction curves showing the number of observed species
when DNA sequence data were rarefied as a function of the number of sequence
reads from the individual macroaggregates and from whole soil. The researchers
describe rarefaction in this way: as you sample sequences from soil, you ask if
you are observing additional species with each additional sequence. For
example, if you had a bowl of M&M's in four colors, you could, in theory,
sample an M&M from the bowl four times and see all four colors. A fifth
time would not add a new color. However, a bowl containing M&M's in a
thousand colors would require much deeper sampling to instill confidence that you
had observed every color. In this case, the researchers observed many hundreds
of species with no evidence that the full diversity of species in whole soil
had been observed.

Acknowledgments:

This research was funded by the Microbial Communities
Initiative, part of PNNL's Laboratory Directed Research and Development (LDRD)
Program, and by ANL's LDRD Program. A
portion of the research was performed at the Environmental Molecular
Sciences Laboratory (EMSL), a national scientific user facility sponsored by the
U.S. Department of Energy (DOE) Office of Biological and Environmental Research
and located at PNNL. Use of
the Advanced Photon Source (APS) at ANL was supported by the DOE Office of
Basic Energy Sciences.